Differential effects of acetyl(C2-) ceramide (N-acetylsphingosine) were studied on coated vesicle formation from Golgi-enriched membranes of Chinese hamster ovary (CHO) and Madin—Darby canine kidney (MDCK) cells. C2-ceramide blocked the translocation of ADP-ribosylation factor-1 (ARF-1) and protein kinase C-α (PKC-α) to the membranes from CHO cells, but not those of MDCK cells. Consequently, C2-ceramide blocked the stimulation of phospholipase D1 (PLD1) by the cytosol and guanosine 5′-[γ-thio]triphosphate (GTP[S]) in membranes from CHO cells. Basal specific activity of PLD1 and the concentration of ARF-1 were 3–4 times higher in Golgi-enriched membranes from MDCK cells compared with CHO cells. Moreover, PLD1 activity in MDCK cells was stimulated less by cytosol and GTP[S]. PLD2 was not detectable in the Golgi-enriched membranes. Incubation of intact CHO cells or their Golgi-enriched membranes with C2-ceramide also inhibited COP1 vesicle formation by membranes from CHO, but not MDCK, cells. Specificity was demonstrated, since dihydro-C2-ceramide had no significant effect on ARF-1 translocation, PLD1 activation or vesicle formation in membranes from both cell types. C2-ceramide also decreased the secretion of virus-like particles to a greater extent in CHO compared with MDCK cells, whereas dihydro-C2-ceramide had no significant effect. The results demonstrate a biological effect of C2-ceramide in CHO cells by decreasing ARF-1 and PKC-α binding to Golgi-enriched membranes, thereby preventing COP1 vesicle formation.
Abbreviations used: ARF, ADP-ribosylation factor; C2-, acetyl; CHO, Chinese hamster ovary; DAG, diacylglycerol; dihydro-C2-ceramide, N-acetyldihydrosphingosine; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; GTP[S], guanosine 5′-[γ-thio]triphosphate; ([3H-methyl]PC), sn-1,2-dipalmitoylglycerophosphoryl[N-methyl-3H]choline; MDCK, Madin—Darby canine kidney; MEM-α, minimum Eagle's medium without nucleosides; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PIP2, phosphatidylinositol 4,5-bisphosphate; PKC, protein kinase C; PLD, phospholipase D; TNFα, tumour necrosis factor-α.
Present address: Laboratoire de Lipolyse Enzymatique, UPR-9025 du CNRS, 31, Chemin Joseph Aiguier, 13402 Marseille, Cedex 20, France.